Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium
| Autor: | Mohammad S, Eram, Benozir, Sarafuddin, Frank, Gong, Kesen, Ma |
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| Rok vydání: | 2015 |
| Předmět: |
BCAA
branched chain amino acid CCE crude cell extract TPP thiamine pyrophosphate IB inclusion body Acetohydroxyacid synthase Thermotogales HTCCE heat-treated crude cell extract Branched-chain amino acids CFE cell-free extract AHAS acetohydroxyacid synthase IMAC immobilized metal affinity chromatography ilv isoleucine leucine valine bacteria Hyperthermophiles TmAHAS Thermotoga maritima acetohydroxyacid synthase health care economics and organizations Research Article |
| Zdroj: | Biochemistry and Biophysics Reports |
| ISSN: | 2405-5808 |
| Popis: | Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent Km and Vmax for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far. Graphical abstract Highlights • First report of AHAS from a hyperthermophilic bacterium. • Catalytic and regulatory subunits of AHAS of T. maritima was expressed in E. coli. • Recombinant proteins were purified using a simplified procedure. • Enzyme represents the most active and thermostable AHAS reported so far. • Kinetic parameters were determined for the purified recombinant enzyme |
| Databáze: | OpenAIRE |
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